Several human skin diseases, including psoriasis and the non-melanoma skin cancers, basal and squamous cell carcinoma, are characterized by excessive growth and aberrant differentiation of epidermal keratinocytes. Some of the signaling pathways that control the temporally and spatially regulated program of keratinocyte differentiation are known; however, the role of others is much less apparent. Previously, we obtained data demonstrating a correlation between keratinocyte differentiation and activation of phospholipase D (PLD), an enzyme that metabolizes phosphatidylcholine to yield phosphatidic acid and choline. However, PLD can also catalyze a transphosphatidylation reaction, in the presence of small amounts of primary alcohols, to generate phosphatidylalcohols. Although this characteristic has been exploited by researchers to monitor PLD activity via alterations in novel phosphatidylalcohols, such as phosphatidylethanol and -butanol, we reasoned that PLD retained this activity throughout the evolutionary process in order to utilize a physiological alcohol. Our subsequent studies demonstrated an association between the glycerol channel, aquaporin-3 (AQP3) and PLD2, as well as an ability of PLD to produce phosphatidylglycerol (PG) in a manner apparently regulated at the level of both the glycerol substrate availability and PLD activity. Based on these results and our preliminary data, we have hypothesized that the PLD2/AQP3/glycerol/PG signaling module represents a novel lipid signaling pathway involved in regulating keratinocyte function. Further, we hypothesize that PG, produced by PLD from the glycerol provided by co- localized AQP3, functions through PKC-Bn, a known PG-responsive kinase expressed in keratinocytes, or another effector enzyme(s) to mediate early keratinocyte differentiation. These hypotheses have the advantage of explaining reports in the literature of a significant skin phenotype observed in AQP3 null mutant and asebia mouse models in association with reduced epidermal glycerol content, as well as the ability of PLD to catalyze the transphosphatidylation reaction. We intend to test the validity of these hypotheses using a variety of approaches, including genetic mouse models, manipulations of protein expression, mass spectrometric analyses of lipid signals, PG overlay assays, pharmacologic inhibitors, activity assays and confocal immunofluorescence studies. By defining the function of this novel PLD2/AQP3/glycerol/PG signaling module in keratinocyte differentiation, this research may identify new potential targets for therapeutic intervention in human skin diseases such as psoriasis and the non- melanoma skin cancers.